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Protein Science (2003), 12:2482-2491.
Copyright © 2003 The Protein Society

Soluble mimics of a chemokine receptor: Chemokine binding by receptor elements juxtaposed on a soluble scaffold

Amita Datta and Martin J. Stone

Department of Chemistry, Indiana University, Bloomington, Indiana 47405-0001, USA

Reprint requests to: Martin J. Stone, Department of Chemistry, Indiana University, Bloomington, IN 47405-0001, USA; e-mail: mastone{at}indiana.edu; fax: (812) 855-8300.

(RECEIVED June 12, 2003; FINAL REVISION July 23, 2003; ACCEPTED July 24, 2003)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03254303.


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Despite the broad biological importance of G protein–coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N-terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 ± 0.8 µM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.

Keywords: Chemokine; chemokine receptor; G protein-coupled receptor; protein design; protein chimera; protein-protein interactions


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
G protein–coupled receptors (GPCRs) transmit signals across biological membranes in response to numerous proteins, peptides, or small molecules (Baldwin 1994; Gudermann et al. 1996). The topology of GPCRs has been deduced by phylogenetic analysis and confirmed with the crystal structure of the photoreceptor bovine rhodopsin (Palczewski et al. 2000). GPCRs consist of a single polypeptide chain that contains seven membrane-spanning helical segments (Baldwin 1994; Gudermann et al. 1996; Fig. 1Go). The cytoplasmic elements associate with heterotrimeric G proteins, whereas the extracellular (and possibly transmembrane) elements are involved in ligand recognition (Baldwin 1994). However, a detailed understanding of ligand recognition by GPCRs remains elusive because of the difficulty isolating or crystallizing these integral membrane proteins (Strader et al. 1994). One approach to circumventing these problems is to develop soluble proteins that mimic the ligand-binding elements of GPCRs. We report the design, preparation, and characterization of a soluble protein in which two ligand-binding elements of the GPCR CC chemokine receptor 3 (CCR3) are displayed on the surface of a soluble protein.



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  		Figure 1. Design and sequences of CROSS proteins. (A) Schematic representation of the CROSS protein design. (Left) Predicted topology of chemokine receptors, showing the membrane-spanning helices (cylinders), N-terminal region (N, red), extracellular loops (E1 and E2, black; E3, red), and disulfide bonds (double lines). (Center) B1 domain of protein G (Protein Data Bank code 2GB1 [PDB] ), showing the {alpha}-helix (cylinder) and ß-strands (arrows) and highlighting the ß2-{alpha}-helix turn (green). (Right) CROSS protein, showing the chemokine receptor elements (red), displayed on the B1 domain scaffold. The N-terminal region of the chemokine receptor is attached at the N terminus of the B1 domain via a (Gly)3 linker (blue), whereas the E3 loop of the chemokine receptor is inserted between residues Ala-20 and Ala-23 of the ß2-{alpha}-helix turn (green), with (Gly)2 linkers (blue) at each junction. (B) CROSS protein sequences. The sequence of each CROSS variant is aligned with CROSS1. Dashes indicate the corresponding CROSS1 residues, and dots indicate gaps in the sequence. The CROSS protein controls containing only the N terminus or E3 loop of CCR3 are designated CROSS-N3 and CROSS-E33, respectively. The color-coding is indicated in A. Cysteine residues in the CCR3 regions of the CROSS proteins are bolded in black. In the CROSS control proteins, these cysteine residues are replaced with serine residues to prevent protein oligomerization.

 
Chemokine/receptor interactions are critical in regulating tissue infiltration by leukocytes, a central feature of inflammatory responses (Baggiolini et al. 1997). Chemokine receptors are differentially expressed on different classes of leukocytes, and chemokines (small proteins) are differentially expressed in various healthy or inflamed tissues (Baggiolini et al. 1997); these expression patterns, and the specificity of chemokine/receptor recognition, control the tissue distribution of leukocytes in normal host defenses and in tissue inflammation (Baggiolini et al. 1997). In addition, chemokine receptors are required for the infection of leukocytes by human immunodeficiency virus (HIV-1) and this receptor function is suppressed by chemokine binding (Cocchi et al. 1996; Feng et al. 1996). A detailed understanding of chemokine/receptor recognition would clearly facilitate efforts to develop specific anti-inflammatory or HIV-suppressive agents. In this work, we used the complex between CCR3 and its cognate chemokine eotaxin to demonstrate the feasibility of preparing a soluble GPCR analog. CCR3 is the most abundant chemokine receptor on eosinophils (Daugherty et al. 1996); activation of CCR3 by eotaxin leads to eosinophil recruitment in allergic asthma, allergic rhinitis, atopic dermatitis, and gastroenteritis (Griffiths-Johnson et al. 1993; Rothenberg et al. 1997).

There is biochemical and biophysical evidence that the extracellular elements of CCR3 play an important role in eotaxin binding. An isolated peptide corresponding to the N-terminal extracellular element of CCR3 binds to eotaxin with 80 ± 40 µM affinity (Ye et al. 2000). Although linear or cyclic peptides corresponding to the three extracellular loops of CCR3 (designated E1, E2, and E3) do not bind in isolation to eotaxin, chimeras of CCR3 with the chemokine receptor CCR1 indicate that the N-terminal segment and the E3 loop of CCR3 (Fig. 1AGo) both participate in eotaxin recognition (Pease et al. 1998). These two elements are predicted to be linked by a disulfide bond (Blanpain et al. 1999). These results indicated that the interaction of the E3 loop with eotaxin may require the structural context of CCR3 and/or may be cooperative with the interactions of other receptor elements. The soluble receptor analogs reported herein incorporate both the N-terminal and E3 segments of CCR3. These receptor mimics are designated by the acronym CROSS (chemokine receptor elements on a soluble scaffold). We describe the design and expression of CROSS proteins and the characterization of their interactions with eotaxin.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Design considerations and choice of scaffold
We designed our soluble receptor mimic (Fig. 1AGo) to satisfy the following criteria: (1) It should incorporate the N-terminal and E3 segments of CCR3; (2) these elements should be displayed on the surface of a folded soluble protein (the scaffold protein); (3) the relative orientation (handedness) of the two receptor elements should be that predicted for the native receptor (Daugherty et al. 1996; Palczewski et al. 2000); (4) there should be a disulfide bond between the two receptor elements, as predicted for CCR3 (Blanpain et al. 1999); and (5) there should be flexible linkers between the scaffold and the binding elements that allow these regions to adopt the most favored ligand-binding conformation without an excessive decrease in entropy upon ligand-binding.

There are a variety of different proteins that could potentially be used as scaffolds in the above design. In the current work, we chose the B1 domain of Streptococcal protein G (Fig. 1AGo). The B1 domain is small (~6 kD), is extremely thermostable ({Delta}Gunfolding = 28 kJ/mole at 30°C; Tm = 89°C), and has been studied extensively by physical and biochemical methods (Achari et al. 1992; Barchi et al. 1994; Gronenborn et al. 1996). Knowledge of the B1 domain’s structure (Clore and Gronenborn 1992; Gallagher et al. 1994), dynamics (Alexander et al. 1992a,b; Barchi et al. 1994; Blanco and Serrano 1998), and residues critical for folding and stability (Kuszewski et al. 1994; Orban et al. 1995; Gronenborn et al. 1996; Sheinerman and Brooks 1998) made it an ideal choice as the scaffold. Herein, we describe the preparation and characterization of several CROSS proteins in which the N-terminal and E3 elements of CCR3 are displayed on the surface of the B1 domain or variants thereof (Fig. 1AGo). The different CROSS proteins are distinguished from each other by a superscripted suffix; for example, CROSS1 is the first CROSS protein prepared.

Preparation and characterization of CROSS1
The initial CROSS protein was constructed by attachment of the N-terminal segment of CCR3 to the N-terminal end of the B1 domain, via a (Gly)3 linker, and insertion of the E3 loop of CCR3 into the ß2-{alpha}-helix turn of the B1 domain (between residues Ala-20 and Ala-23), with (Gly)2 linkers at each junction (Fig. 1AGo). The glycine-rich linkers are intended to provide conformational flexibility and to approximately align the ends of the receptor elements.

CROSS1 was expressed in Escherichia coli as a fusion protein with an N-terminal His6-tag. The fusion protein was expressed in inclusion bodies, purified under denaturing conditions, and dialyzed into native buffer. Proteolytic removal of the His6-tag followed by ion exchange chromatography yielded homogeneous CROSS1 with the expected mass (13,003.5 Da found; 13,000.3 Da calculated). Nonreducing SDS-PAGE and N-ethyl maleimide (NEM) derivatization (monitored by MALDI-TOF [matrix-assisted laser-desorption ionization time-of-flight] mass spectrometry) indicated that CROSS1 is monomeric and that the disulfide bond between the two CCR3 elements is formed. However, the far-UV circular dichroism (CD) spectrum of CROSS1 (Fig. 2AGo) indicated that CROSS1 is predominantly unfolded under native conditions. We attempted to induce folding of CROSS1 by addition of osmolytes; this strategy has been effective for a variety of other proteins (Bhattacharjya and Balaram 1997; David-Searles et al. 1998; Vuillard et al. 1998; Baskakov et al. 1999). Glucose, sucrose, trehalose, stachyose, and trimethylamine oxide all increased the negative dichroism at 222 nm. The largest increase was observed for sucrose, although very high sucrose concentrations (>0.5 M) were required (Fig. 2AGo).



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  		Figure 2. Characterization of CROSS1. (A) The far-UV CD spectra of the B1 domain (solid line), CROSS1 in the absence of sucrose (open circles), and CROSS1 in the presence of 700 mM sucrose (open squares). All spectra were recorded using 20 µM protein in 20 mM NaH2PO4 and 150 mM NaCl (pH 8.0) at 4°C. (B, C) SPR sensorgrams of CROSS1 in the absence and the presence of 700 mM sucrose, respectively. Each curve is an average of three measurements at the CROSS1 concentration indicated. (D) Equilibrium binding curves, measured using SPR for interactions of CROSS1 and CCR3(1-35) with biotin-labeled eotaxin. Titration of biotin-eotaxin with CROSS1 in the absence (open circles) and the presence (open squares) of 700 mM sucrose, and with CCR3(1-35) in the absence (filled circles) and the presence (filled squares) of 700 mM sucrose.

 
The affinity of CROSS1 for biotinylated eotaxin, immobilized on a streptavidin-coated chip, was measured by using an equilibrium surface plasmon resonance (SPR) assay (Fig. 2BGo–D); measurement of the binding kinetics was not possible due to fast dissociation of the complex. CROSS1 bound to eotaxin with an equilibrium dissociation constant (Kd) of 64 ± 16 µM in the absence of sucrose, comparable to the affinity of eotaxin for the isolated N-terminal fragment of CCR3, designated CCR3(1-35) (Kd = 120 ± 36 µM by SPR and 80 ± 40 µM by NMR). However, in the presence of 700 mM sucrose, the Kd of CROSS1 for eotaxin decreased to 23 ± 9 µM, whereas the Kd of the CCR3(1-35) for eotaxin did not change significantly (Kd = 154 ± 23 µM by SPR). These results (summarized in Table 1Go) indicated that inducing a native-like structure in the B1 domain core of CROSS1 may bring the two CCR3 elements of CROSS1 sufficiently close to allow simultaneous interaction with eotaxin. Thus, we undertook the design of additional CROSS proteins in which the scaffold was more likely to fold under native conditions.


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  		Table 1. Eotaxin binding affinities of CROSS proteins and controls
 
Stabilization of the B1 domain core: CROSS4
Two groups have previously reported hyperstabilized B1 domain mutants with substantially enhanced stability (Tm > 100°C) compared with the wild-type B1 domain. Baker and coworkers identified two unrelated ß1-ß2 hairpin turn sequences that give rise to B1 domain variants with greater intrinsic stability than did the wild-type domain (D.A. Baker, pers. comm.). Malakauskas and Mayo (1998) used a computational algorithm to design a hyperstable B1 domain mutant with multiple mutations, mainly in the hydrophobic core. In an attempt to obtain a CROSS protein that was folded in the absence of osmolytes, we replaced the B1 domain core of CROSS1 with B1 domain variants containing the Baker ß-hairpin sequences (CROSS2 and CROSS3) or the Mayo hydrophobic core (CROSS4; Fig. 1BGo). All three were expressed in E. coli inclusion bodies and purified similarly to CROSS1, and all three gave CD spectra indicative of enhanced secondary structure, although the helical content of CROSS4 appears to be the highest (Fig. 3AGo). CROSS2 and CROSS3 had a tendency to aggregate, indicating that they might form nonnative oligomeric structures, as was observed recently for a hydrophobic core mutant of the B1 domain (Frank et al. 2002). CROSS4 remained soluble, could be readily characterized, had the expected molecular weight (13,072.4 Da found; 13,071.5 Da calculated), and contained the disulfide bond between N-terminal and E3 elements.



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  		Figure 3. Characterization of CROSS2, CROSS3, and CROSS4. (A) The far-UV CD spectra of the B1 domain (solid line), CROSS2 (open circles), CROSS3 (open squares), and CROSS4 (open triangles). All spectra were recorded using 20 µM protein in 20 mM NaH2PO4 and 150 mM NaCl (pH 8.0) at 4°C. (B) SPR equilibrium assay sensorgram of CROSS4. (C) SPR kinetic assay sensorgram of CROSS4. Curves shown in B and C are averages of three measurements at the CROSS4 concentrations indicated. (D) Equilibrium binding curves, measured using SPR, for interactions of CROSS4 (open circles), CROSS4-E33 (filled circles), and CROSS4-N3 (filled squares) with biotin-labeled eotaxin, and an equilibrium binding curve for interaction of CROSS4 with fluorescein-labeled eotaxin (open squares). The inset shows the CROSS4 control data over a broader protein concentration range.

 
The affinity of CROSS4 for eotaxin was determined by the equilibrium SPR assay to be 4.2 ± 1.6 µM, ~15- to 25-fold tighter than binding of either CROSS1 or CCR3(1-35) to eotaxin (Fig. 3B,D). To test whether both the N-terminal and E3 elements of CCR3 were contributing to this interaction, we prepared two control proteins from which each of these elements was removed. Removal of the N-terminal segment increased the Kd to 72 ± 26 µM, whereas removal of the E3 segment increased the Kd to 131 ± 18 µM, providing evidence for the involvement of both elements in the binding interaction (Table 1Go). The B1 domain scaffold showed no interaction with eotaxin in the SPR experiment.

We have corroborated the higher affinity of CROSS4 for eotaxin by using two additional binding methods. First, the kinetics of binding were determined by SPR, yielding kon = 7.89 x 103 ± 187 M-1sec-1, koff = 3.21 x 10-2 ± 2.68 x 10-4 sec-1, and Kd = koff/kon = 4.0 ± 0.1 µM (Fig. 3CGo). Second, eotaxin was labeled with fluorescein, and the fluorescence anisotropy was determined as a function of CROSS4 concentration, yielding a Kd value of 3.6 ± 0.8 µM (Figs. 3D, 4BGoGo). There is excellent agreement among the three methods used. The fluorescence anisotropy technique was used for subsequent measurements because it is a measure of the binding equilibrium in solution rather than on a solid support.



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  		Figure 4. Characterization of CROSS5, CROSS6, and CROSS7. (A) The far-UV CD spectra of the B1 domain (solid line), CROSS4 (solid squares), CROSS5 (open circles), CROSS6 (open squares), and CROSS7 (open triangles). All spectra were recorded using 20 µM protein in 20 mM NaH2PO4, 150 mM NaCl (pH 8.0) at 4°C. (B) Titration of fluorescein-labeled eotaxin with CROSS4 (filled diamonds), CROSS5 (open circles), CROSS6 (open squares), CROSS7 (open triangles), CROSS5-N3 (filled circles), and CROSS5-E33 (filled squares). Errors in anisotropy are comparable to the size of the symbols shown. (Inset) CROSS5 data over a broader protein concentration range.

 
Introduction of an additional disulfide bridge: CROSS5
In an attempt to further stabilize the CROSS scaffold, we introduced each of three disulfide bonds between adjacent ß-strands in the stabilized B1 domain core of CROSS4. CROSS5 has a disulfide between B1 domain residue 2 in the first ß-strand (ß1) and residue 19 in the second ß-strand (ß2); CROSS6 has a disulfide between residue 6 in the first ß-strand (ß1) and residue 53 in the fourth ß-strand (ß4); and CROSS7 has a disulfide between residue 8 in the first ß-strand (ß1) and residue 55 in the fourth ß-strand (ß4). Fortuitously, CROSS5, CROSS6, and CROSS7 were all expressed as soluble proteins in E. coli. After native Ni-affinity chromatography, removal of the His6-tag, and ion exchange chromatography, homogeneous proteins with the expected molecular weights were obtained, with CROSS5 being obtained in the highest yield (~10 to 15 mg/L). The CD spectra indicated secondary structure contents at least as high as that of CROSS4, but substantially higher in the case of CROSS5 (Fig. 4AGo). CROSS5, CROSS6, and CROSS7 all bind to eotaxin with affinities of ~3 µM, as determined by fluorescence anisotropy (Fig. 4BGo; Table 1Go). Removal of the N-terminal segment from CROSS5 increased the Kd to 135 ± 48 µM, whereas removal of the E3 segment increased the Kd to 179 ± 108 µM, again supporting the contention that both elements participate in the binding interaction (Fig. 4BGo; Table 1Go). The interaction of CROSS5 with eotaxin was characterized in more detail.

Comparison of CROSS5 recognition with CCR3 recognition by eotaxin
To determine whether CROSS5 interacts with the same regions of eotaxin that are recognized by CCR3, we tested the ability of CROSS5 to compete with CCR3 for binding to eotaxin. CROSS5 inhibits the binding of 125I-labeled eotaxin to CCR3 expressed on L1.2 murine pre-B cells, with a 50% inhibitory concentration (IC50) of 7.9 ± 1.4 µM (Fig. 5AGo), demonstrating that CROSS5 and CCR3 have overlapping binding sites on eotaxin. The small difference between the observed in vitro Kd and in vivo IC50 values may be due to the different buffer conditions used for the two assays and/or nonspecific association of eotaxin (or possibly CROSS5) with cell surface molecules such as glycosaminoglycans (GAGs); GAG-binding to chemokines is well documented (Hoogewerf et al. 1997; Chakravarty et al. 1998; Ali et al. 2000; Laurence et al. 2001).



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  		Figure 5. Comparison of CROSS5 recognition with CCR3 recognition by eotaxin. (A) Inhibition of 125I-eotaxin binding to CCR3 on murine pre-B cells (L1.2) by increasing concentrations of CROSS5. The average and the standard deviation of duplicate measurements are shown. The solid line is a fit to the competitive binding equation given in the Materials and Methods section. (B) Competitive binding of CROSS5 to fluorescein-labeled wild-type eotaxin and representative unlabeled chemokines (wild-type and five eotaxin mutants). Increasing concentrations of unlabeled chemokines displace the labeled eotaxin from CROSS5, hence decreasing the observed anisotropy. Data are shown for wild-type eotaxin (open circles), R16A (open squares), I18A (open triangles), V5A (filled circles), S4A (filled squares), and F11A (filled triangles). Solid lines are fits to theoretical binding isotherms (see Materials and Methods). (C) Correlation of the apparent affinities (IC50) of wild-type eotaxin and 12 eotaxin mutants for CCR3 with the affinities (Kd) for CROSS5. Wild-type eotaxin (WT), R16A, and K17A are labeled. Other points are for the mutants as follows: S4A, V5A, T7A, T8A, F11A, N12A, L13A, N15A, I18A, and L20A. Kd values were determined by competitive fluorescence anisotropy, as shown in B, and IC50 values are from Mayer and Stone (2001).

 
As an additional assessment of the similarity between eotaxin binding to CROSS5 and CCR3, we compared the binding affinities of 12 eotaxin mutants to both proteins. The mutations are spread throughout the first 20 residues of eotaxin, spanning the N-terminal (residues 1–10) and N-loop regions (residues 11–19), both of which have been implicated in receptor recognition (Mayer and Stone 2001). The affinities (Kd values) of unlabeled wild-type and mutant eotaxin for CROSS5 were determined by monitoring their ability to displace fluorescein-labeled eotaxin from CROSS5, using fluorescence anisotropy (Fig. 5BGo). The apparent affinities of the same species for CCR3 have been determined previously by using a competitive radioligand-binding assay (Mayer and Stone 2001). There is a strong correlation between affinity for CROSS5 and apparent affinity for CCR3 (r = 0.88, Fig. 5CGo), indicating that the chemokine elements that contribute to CROSS5 binding make similar free energy contributions to CCR3 binding. The only exceptions are the R16A and K17A mutants, which each bind more tightly to CROSS5 than would be expected based upon their CCR3 affinities. Thus, R16A and K17A, in the N-loop region of eotaxin, may interact with elements of CCR3 not included in CROSS5. Another possibility is that these basic residues are involved in binding negatively charged cell surface GAGs, thereby improving their apparent CCR3 binding affinities. Recent reports indicate that basic residues within chemokines interact with GAGs, resulting in increased local concentrations of chemokine oligomers and promotion of leukocyte chemotaxis in vivo (Laurence et al. 2001; Proudfoot et al. 2003). Omission of R16A and K17A from Figure 5CGo improves the correlation coefficient to 0.97.


   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
The extracellular domains of GPCRs have been implicated as important elements for ligand recognition by using a variety of experimental approaches, including analyses of receptor mutants (Leong et al. 1994) and chimeras (Monteclaro and Charo 1996Monteclaro and Charo 1997; Wu et al. 1996; Samson et al. 1997; Pease et al. 1998), cross-linking of labeled ligands to receptors (Baldwin 1994), binding of ligands to peptide fragments from receptors, and molecular modeling (Luo et al. 1997). Herein, we report an additional approach, the binding of a GPCR ligand by two receptor elements juxtaposed on a soluble protein scaffold. The receptor mimic CROSS5 binds to the ligand eotaxin with moderate affinity (Kd ~ 3 µM). The ability of CROSS5 to compete with CCR3 for binding to eotaxin indicates that the binding site of the receptor analog overlaps that of the natural receptor. Furthermore, there is a strong correlation between the affinities of several eotaxin mutants for CROSS5 and the apparent affinities of the same mutants for CCR3. The observation that mutated residues make similar free energy contributions to binding either CROSS5 or CCR3, strongly indicates that the structural basis of CROSS5 recognition is similar to that of the natural receptor.

In the course of developing CROSS5, we surveyed a variety of CROSS proteins and controls. Comparison of the properties of these proteins provides some insights into the structural requirements for eotaxin recognition. First, control proteins lacking either the N-terminal or E3 element bind 34- to 45-fold more weakly than does CROSS5 to eotaxin, demonstrating the involvement of both the CCR3 N-terminal and E3 elements in the interaction. Second, the only difference between CROSS1 and CROSS4 is the structure of the scaffold, the latter being folded under native conditions. However, CROSS4 binds ~16-fold more tightly than does CROSS1 to eotaxin. Thus, a folded scaffold is required to position the two CCR3 elements appropriately for simultaneous interactions with the ligand. Taken together, the comparisons of different CROSS proteins and the mutant analysis discussed above indicate that the relative placement and orientation of the CCR3 elements within the CROSS/eotaxin complexes must be similar to those in the natural CCR3/eotaxin complex.

The substantial affinities observed for the CROSS proteins raise the question as to whether CCR3 uses only the N-terminal and E3 elements for high-affinity binding of eotaxin. The mutant correlation analysis (Fig. 5CGo) indicates most of the interactions between CCR3 and the mutated regions of eotaxin (residues 4–20) involve the regions of CCR3 that are present in CROSS5. Nevertheless, the affinity of CROSS5 for eotaxin is approximately three orders of magnitude lower than the apparent affinity of eotaxin for CCR3 (Kd,app ~ 1 to 2 nM on whole cells; Mayer and Stone 2001). There are several possible reasons for this difference. First, eotaxin may interact with additional elements of CCR3. In support of this possibility, the R16A and K17A mutations of eotaxin decrease the affinity for CCR3 by 10- and 12-fold, respectively, but decrease the affinity for CROSS5 by only 2-fold and 1.9-fold, respectively (Fig. 5CGo). In addition, mutants and/or chimeras of several chemokine receptors indicate roles for the E1 and/or E2 loops in chemokine binding (Monteclaro and Charo 1996Monteclaro and Charo 1997; Samson et al. 1997; Pease et al. 1998; Ye et al. 2000). If additional elements are involved in eotaxin recognition, then incorporation of these elements into soluble model systems may yield soluble receptor analogs with higher affinity than CROSS5 for eotaxin. A second possible explanation of the lower affinities of CROSS5 compared with CCR3 is that the structures and/or dynamics of the receptor elements in CROSS5 may differ from those of the corresponding elements in the natural receptor. In this regard, alternative scaffolds and/or alternative linker sequences could potentially enhance the affinity of soluble receptor mimics for chemokines; a combinatorial approach such as phage display may prove useful for these alterations. A third possibility is that high affinity binding of CCR3 may require posttranslational modifications that are not present in CROSS5. In particular, the N termini of chemokine receptors are known to be sulfated on tyrosine residues, and sulfation appears to be required for ligand binding and/or activity (Farzan et al. 1999).

Although we know of no precedent for the development of soluble GPCR receptor analogs, the study of tyrosine kinase receptors (TKRs) and the glutamate receptor (GR) has been extensively facilitated by soluble receptor mimics. In TKRs, the extracellular region is an autonomously folded domain (Postel-Vinay 1996; Zhan et al. 1999; Hoyne et al. 2000), whereas in GR there are two extracellular ligand-binding domains, and the intervening transmembrane region can be replaced by a hydrophilic linker peptide (Kuusinen et al. 1995). For both TKRs and GR, the soluble mimics have been co-crystallized with the cognate ligands, leading to a dramatically improved understanding of receptor recognition and function (Zhan et al. 1999; Hogner et al. 2002). Clearly, structural studies of the CROSS protein complexes described herein have the potential to reveal the molecular details of eotaxin/CCR3 interactions. Moreover, similar receptor analogs could potentially be developed for other GPCRs in which the extracellular regions are the major ligand-recognition elements, including the receptors for other chemokines (Howard et al. 1996), peptide hormones (Strader et al. 1994), and complement (Boulay et al. 1997). Finally, one can imagine a variety of other applications of CROSS proteins, including mutational analysis to identify receptor elements involved in ligand recognition; biochemical procedures for the affinity purification, detection, and analysis of ligands; inhibition of receptor biochemical or biological activity; and the screening of drug candidates that potentially inhibit receptor binding.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Protein preparation
Chemokines were expressed in E. coli and purified as described (Paavola et al. 1998; Ye et al. 2000; Mayer and Stone 2001). Fluorescein- and biotin-labeled chemokines for anisotropy measurements were obtained by reaction of single cysteine mutants with 5-idoacetamidofluorescein (Molecular Probes) or biotin-HPDP (Pierce Chemical Co.); mutations were near the C termini and did not influence affinities for receptors or CROSS proteins. The codon-optimized CROSS1 gene was synthesized by recursive PCR and subcloned into the expression vector pET-28a (Novagen, Inc.). Genes encoding the other CROSS proteins were obtained by modification of the CROSS1 construct. All CROSS proteins were overexpressed as N-terminal His6-tag fusion proteins in BL21(DE3) E. coli by using the T7 promoter system. Cells were grown at 37°C in Luria Broth supplemented with 30 µg/mL kanamycin to an OD600 of 0.7 to 0.8, and expression was induced with 1 mM IPTG (Sigma Chemical Co.). After induction, the cells were grown for 12 to 16 h at 25°C and then harvested by centrifugation for 10 min at 5000g at 4°C. For the purification of CROSS5, CROSS6, and CROSS7, cells were lysed in buffer (50 mM Na2HPO4, 500 mM NaCl, and 5 mM imidazole at pH 8.0) with hen egg white lysozyme (1 mg/mL; Sigma Chemical Co.) and subjected to three freeze/thaw cycles by using liquid nitrogen. Lysate was then sonicated on ice at 60% power by using 30-sec bursts for a total of 10 min with 2 min of incubation on ice between each burst. Cell lysate was cleared by centrifugation at 15,000g for 30 min at 4°C. The cleared supernatant was loaded onto a Ni-NTA affinity column (Qiagen) equilibrated with lysis buffer at 0.5 mL/min. The column was washed with a gradient of buffers containing up to 30 mM imidazole in 50 mM Na2HPO4 and 500 mM NaCl at 1.0 mL/min until the OD280 was <0.05. CROSS was eluted with 50 mM Na2HPO4, 500 mM NaCl, and 200 mM imidazole (pH 8.0) at 1.0 mL/min. After elution from the Ni-NTA column, the N-terminal His6-tag was removed by incubation of CROSS with human thrombin (Sigma Chemical Co.; 1 µg/mg fusion protein) overnight at room temperature in 20 mM Tris-HCl (pH 8.0). The mature protein was then purified on a HiTrap Q Sepharose anion exchange chromatography column (Pharmacia Biotech) by using 20 mM bis-Tris (pH 6.5) containing a gradually increasing gradient of NaCl (20 mM/min over 120 min at a flow rate of 0.8 mL/min). Homogeneous CROSS5, CROSS6, and CROSS7 eluted as a major peak at 200 to 250 mM NaCl. The typical yield of a 1 L culture was 2 to 3 mg of pure protein. The earlier generation CROSS proteins were purified similarly to CROSS5, except that (1) they were isolated from purified inclusion bodies; (2) the inclusion bodies were solubilized by using 6 M GnHCl, 0.1 M NaH2PO4, and 0.01 M TrisHCl (pH 8.0); (3) Ni-NTA affinity chromatography was performed under denaturing conditions; and (4) a refolding step was included before thrombin cleavage. For each protein, the MALDI-TOF mass spectrum was consistent with the predicted monomer molecular weight. CROSS protein reaction with NEM was carried out as described previously (Ye et al. 2000).

Secondary structure determination
CD spectra were recorded on a Jasco J-715 spectropolarimeter. Each spectrum is the sum of five scans recorded at 20 nm/min and a resolution of 1 nm by using a 0.1-cm cuvette at 4°C. Control scans were recorded under the same parameters as ones containing protein sample. The control values were subtracted from the scans of the protein samples to eliminate background signal resulting from the buffer.

Surface plasmon resonance
Coupling of biotinylated eotaxin to the gold surface of the sensor chip was achieved by flowing 5 µM biotinylated eotaxin in a 10 mM Hepes, 100 mM NaCl, and 0.05% Tween 20 (pH 7.4) buffer over a streptavidin-coated (SA) sensor chip (Biacore Inc.). The amount of biotinylated eotaxin immobilized was determined after washing to be 200 RU for CROSS4 kinetic assays and 5500 RU for CROSS1, CCR3(1-35), CROSS4, and CROSS4 control protein equilibrium assays. A control surface was prepared by flowing free biotin (0.003 mg/mL) over a second flow cell of the SA sensor chip, and data from this "blank" cell were subtracted from the sample data. Sensorgrams were collected for all proteins (at the concentrations indicated in Results) flowed over a sensor chip containing SA-biotin-eotaxin in 10 mM Hepes, 150 mM NaCl, 3mM EDTA, and 0.005% (v/v) Tween 20 (pH 7.4) at 4°C.

Kinetic SPR experiments for CROSS4 were carried out at a flow rate of 20 µL/min and a sampling rate of 1 Hz on a Biacore 3000. Kinetic binding constants for the eotaxin/CROSS4 interaction were determined from data recorded at five different CROSS4 concentrations by using the program BIAevaluation, version 3.0. Global fits were determined from an average of three data sets collected on separate days by using different protein preparations. Curve fits for both association and dissociation phases of the sensorgram showed low {chi}2 values and low residuals.

CROSS1, CCR3(1-35), CROSS4, and CROSS4 control protein equilibrium SPR assays were carried out at a flow rate of 5 µL/min and a sampling rate of 1 Hz on a Biacore 3000. Sensorgrams from three sets of data for each of the protein concentrations were collected and averaged for each protein. The Kd values were obtained by fitting data to the Langmuir binding model by using the Sigma Plot software version 4.0 (SPSS Science), according to the following equation: 1/Kd = Req/C(Rmax - Req), where Rmax is the total surface binding capacity in RU, Req is the steady state binding level in RU, and C is the CROSS or CCR3(1-35) concentration.

Fluorescence anisotropy
Anisotropy measurements of fluorescein-labeled chemokines were recorded at 4 °C on a Perkin Elmer LS-50b fluorometer by using excitation and emission wavelengths of 494 and 518 nm, respectively. Samples were dissolved in 10 mM Hepes, 150 mM NaCl, and 3 mM EDTA (pH 7.4). For CROSS protein binding to fluorescein-labeled wild-type chemokines, duplicate affinity measurements were made by titration of CROSS proteins into 100 nM solutions of the chemokines, and data were fit to a single-site binding isotherm. For the competitive displacement of fluorescein-labeled eotaxin (FITC-eotaxin) from CROSS5 with unlabeled chemokines, the FITC-eotaxin was premixed with CROSS5 to 75% saturation, and increasing amounts of unlabeled chemokine were added. Displacement curves were fit as described (Huff et al. 2003) to yield the Kd of CROSS5 for each unlabeled chemokine.

125I-eotaxin binding assay
The competitive binding assay was performed in duplicate as previously reported using the murine L1.2 pre-B cell line transfected with CCR3 (Mayer and Stone 2001). Briefly, 5 x 106 L1.2-CCR3 cells/mL in 25 mM HEPES, 1nM CaCl2, 120 mM NaCl, and 0.5% bovine serum albumin (protease-free; pH 7.6) were incubated with ~0.15 nM 125I-eotaxin (~2000 Ci/mmole; Amersham Pharmacia Biotech) and increasing concentrations of CROSS5 in 250 µL for 4.5 h at 4°C. After incubation, the cells were separated from the unbound protein and resuspended, and radioactivity was counted as described previously (Mayer and Stone 2001). The data were fit to the following equation by using SigmaPlot, version 4.0 (SPSS Science): % radioligand bound = 100% - {(L)/(IC50 + [L])}, in which L represents CROSS5, and IC50 is the concentration of CROSS5 required to displace half of the CCR3-bound 125I-eotaxin.


   Acknowledgments
 
We thank Drs. K.L. Mayer, M.R. Mayer, M.G. Oakley, and T.R. Parody for discussions and M.R. Mayer for providing eotaxin mutants. This work was supported by grants to M.J.S. from the NIH (GM 55055) and the American Heart Association (Established Investigator Award) and by fellowships to A.D. from GAANN (Graduate Assistance in Areas of National Need) and the Kraft Foundation.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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